RESUMO
CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.
Assuntos
Cisteína Endopeptidases/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Colite/imunologia , Colite/patologia , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/patologia , Vírus da Coriomeningite Linfocítica/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/citologia , Células Th1/citologiaRESUMO
Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 µm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.
Assuntos
Detecção Precoce de Câncer/métodos , Endoscopia/métodos , Lesões Pré-Cancerosas/diagnóstico , Animais , Colo/patologia , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Feminino , Fluorescência , Corantes Fluorescentes , Neoplasias Gastrointestinais/patologia , Trato Gastrointestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular/métodos , Lesões Pré-Cancerosas/patologia , Ratos , Ratos Endogâmicos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/prevenção & controle , SuínosRESUMO
Fluorescently quenched probes that are specifically activated in the cancer microenvironment have great potential application for diagnosis, early detection, and surgical guidance. These probes are often designed to target specific enzymes associated with diseases by direct optimization using single purified enzymes. However, this can result in painstaking chemistry efforts to produce a probe with suboptimal performance when applied inâ vivo. We describe here an alternate, unbiased activity-profiling approach in which whole tissue extracts are used to directly identify optimal peptide sequences for probe design. Screening of tumor extracts with a hybrid combinatorial substrate library (HyCoSuL) identified a combination of natural and non-natural amino-acid residues that was used to generate highly efficient tumor-specific probes. This new strategy simplifies and enhances the process of probe optimization without any aâ priori knowledge of enzyme targets and has the potential to be applied to diverse disease states using clinical or animal-model tissue samples.
Assuntos
Corantes Fluorescentes/química , Imagem Óptica/métodos , Animais , Técnicas de Química Combinatória , Xenoenxertos , Humanos , Camundongos , Proteólise , Reprodutibilidade dos Testes , Especificidade por Substrato , Extratos de Tecidos/química , Microambiente TumoralRESUMO
Proteases are regulators of diverse biological pathways including protein catabolism, antigen processing and inflammation, as well as various disease conditions, such as malignant metastasis, viral infection and parasite invasion. The identification of substrates of a given protease is essential to understand its function and this information can also aid in the design of specific inhibitors and active site probes. However, the diversity of putative protein and peptide substrates makes connecting a protease to its downstream substrates technically difficult and time-consuming. To address this challenge in protease research, a range of methods have been developed to identify natural protein substrates as well as map the overall substrate specificity patterns of proteases. In this review, we highlight recent examples of both synthetic and biological methods that are being used to define the substrate specificity of protease so that new protease-specific tools and therapeutic agents can be developed.
Assuntos
Peptídeo Hidrolases/genética , Peptídeos/genética , Especificidade por Substrato/genética , Humanos , Peptídeo Hidrolases/química , Peptídeos/antagonistas & inibidores , Peptídeos/química , Inibidores de Proteases/química , Transdução de Sinais/genéticaRESUMO
Molecularly targeted optical contrast agents have the potential to enable surgeons to visualize specific molecular markers that can help improve surgical precision and thus outcomes. Fluorescently quenched substrates can be used to highlight tumor lesions by targeting proteases that are highly abundant in the tumor microenvironment. However, the majority of these and other molecularly targeted optical contrast agents are labeled with reporter dyes that are not ideally matched to the properties of clinical camera systems, which are typically optimized for detection of indocyanine-green (ICG). While a wide range of near-infrared (NIR) dyes are suitable for use with highly sensitive and highly tunable research-focused small animal imaging systems, most have not been evaluated for use with commonly used clinical imaging systems. Here we report the optimization of a small molecule fluorescently quenched protease substrate probe 6QC-ICG, which uses the indocyanine green (ICG) dye as its optical reporter. We evaluated dosing and kinetic parameters of this molecule in tumor-bearing mice and observed optimal tumor over background signals in as little as 90 min with a dose of 2.3 mg/kg. Importantly, the fluorescence intensity of the probe signal in tumors did not linearly scale with dose, suggesting the importance of detailed dosing studies. Furthermore, when imaged using the FDA approved da Vinci Si surgical system with Firefly detection, signals were significantly higher for the ICG probe compared to a corresponding probe containing a dye with similar quantum yield but with a slightly shifted excitation and emission profile. The increased signal intensity generated by the optimal dye and dose of the ICG labeled probe enabled detection of small, flat lesions that were less than 5 mm in diameter. Therefore, 6QC-ICG is a highly sensitive probe that performs optimally with clinical imaging systems and has great potential for applications in optical surgical navigation.
Assuntos
Meios de Contraste/metabolismo , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias/diagnóstico por imagem , Peptídeo Hidrolases/metabolismo , Cirurgia Vídeoassistida/métodos , Animais , Linhagem Celular Tumoral/transplante , Meios de Contraste/administração & dosagem , Meios de Contraste/química , Feminino , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Verde de Indocianina/administração & dosagem , Verde de Indocianina/química , Verde de Indocianina/metabolismo , Neoplasias Mamárias Experimentais/cirurgia , Camundongos , Camundongos Endogâmicos BALB C , Imagem Molecular/métodos , Neoplasias/patologia , Neoplasias/cirurgiaRESUMO
Nematodes use an extensive chemical language based on glycosides of the dideoxysugar ascarylose for developmental regulation (dauer formation), male sex attraction, aggregation, and dispersal. However, no examples of a female- or hermaphrodite-specific sex attractant have been identified to date. In this study, we investigated the pheromone system of the gonochoristic sour paste nematode Panagrellus redivivus, which produces sex-specific attractants of the opposite sex. Activity-guided fractionation of the P. redivivus exometabolome revealed that males are strongly attracted to ascr#1 (also known as daumone), an ascaroside previously identified from Caenorhabditis elegans hermaphrodites. Female P. redivivus are repelled by high concentrations of ascr#1 but are specifically attracted to a previously unknown ascaroside that we named dhas#18, a dihydroxy derivative of the known ascr#18 and an ascaroside that features extensive functionalization of the lipid-derived side chain. Targeted profiling of the P. redivivus exometabolome revealed several additional ascarosides that did not induce strong chemotaxis. We show that P. redivivus females, but not males, produce the male-attracting ascr#1, whereas males, but not females, produce the female-attracting dhas#18. These results show that ascaroside biosynthesis in P. redivivus is highly sex-specific. Furthermore, the extensive side chain functionalization in dhas#18, which is reminiscent of polyketide-derived natural products, indicates unanticipated biosynthetic capabilities in nematodes.
Assuntos
Nematoides/metabolismo , Atrativos Sexuais/metabolismo , Animais , Quimiotaxia/efeitos dos fármacos , Feminino , Espectroscopia de Ressonância Magnética/métodos , Masculino , Metabolômica , Modelos Biológicos , Modelos Químicos , Oxigênio/química , Peroxissomos/metabolismo , Feromônios/metabolismo , Comportamento Sexual Animal/efeitos dos fármacos , Comportamento Sexual Animal/fisiologia , Transdução de SinaisRESUMO
The ascarosides, small-molecule signals derived from combinatorial assembly of primary metabolism-derived building blocks, play a central role in Caenorhabditis elegans biology and regulate many aspects of development and behavior in this model organism as well as in other nematodes. Using HPLC-MS/MS-based targeted metabolomics, we identified novel ascarosides incorporating a side chain derived from succinylation of the neurotransmitter octopamine. These compounds, named osas#2, osas#9, and osas#10, are produced predominantly by L1 larvae, where they serve as part of a dispersal signal, whereas these ascarosides are largely absent from the metabolomes of other life stages. Investigating the biogenesis of these octopamine-derived ascarosides, we found that succinylation represents a previously unrecognized pathway of biogenic amine metabolism. At physiological concentrations, the neurotransmitters serotonin, dopamine, and octopamine are converted to a large extent into the corresponding succinates, in addition to the previously described acetates. Chemically, bimodal deactivation of biogenic amines via acetylation and succinylation parallels posttranslational modification of proteins via acetylation and succinylation of L-lysine. Our results reveal a small-molecule connection between neurotransmitter signaling and interorganismal regulation of behavior and suggest that ascaroside biosynthesis is based in part on co-option of degradative biochemical pathways.
Assuntos
Aminas Biogênicas/metabolismo , Caenorhabditis elegans/metabolismo , Octopamina/química , Agonistas alfa-Adrenérgicos/química , Animais , Comportamento Animal , Cromatografia Líquida de Alta Pressão , Dopamina/metabolismo , Glicosídeos/química , Espectrometria de Massas , Neurotransmissores/metabolismo , Feromônios/metabolismo , Serotonina/metabolismo , Transdução de Sinais , Succinatos/químicaRESUMO
Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become the standard of care in cancer surgeries. One of the key parameters to optimize in contrast agents is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker, as well as the positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased overall signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.
Assuntos
Corantes Fluorescentes , Neoplasias , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Animais , Neoplasias/diagnóstico por imagem , Humanos , Camundongos , Imagem Óptica/métodos , Peptídeo Hidrolases/metabolismo , Polímeros/química , Linhagem Celular Tumoral , Acrilamidas/químicaRESUMO
Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become standard of care in cancer surgeries. One of the key parameters to optimize in contrast agent is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker as well as positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased over-all signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.
RESUMO
In the model organism Caenorhabditis elegans, a family of endogenous small molecules, the ascarosides function as key regulators of developmental timing and behavior that act upstream of conserved signaling pathways. The ascarosides are based on the dideoxysugar ascarylose, which is linked to fatty-acid-like side chains of varying lengths derived from peroxisomal ß-oxidation. Despite the importance of ascarosides for many aspects of C. elegans biology, knowledge of their structures, biosynthesis, and homeostasis remains incomplete. We used an MS/MS-based screen to profile ascarosides in C. elegans wild-type and mutant metabolomes, which revealed a much greater structural diversity of ascaroside derivatives than previously reported. Comparison of the metabolomes from wild-type and a series of peroxisomal ß-oxidation mutants showed that the enoyl CoA-hydratase MAOC-1 serves an important role in ascaroside biosynthesis and clarified the functions of two other enzymes, ACOX-1 and DHS-28. We show that, following peroxisomal ß-oxidation, the ascarosides are selectively derivatized with moieties of varied biogenetic origin and that such modifications can dramatically affect biological activity, producing signaling molecules active at low femtomolar concentrations. Based on these results, the ascarosides appear as a modular library of small-molecule signals, integrating building blocks from three major metabolic pathways: carbohydrate metabolism, peroxisomal ß-oxidation of fatty acids, and amino acid catabolism. Our screen further demonstrates that ascaroside biosynthesis is directly affected by nutritional status and that excretion of the final products is highly selective.
Assuntos
Caenorhabditis elegans/metabolismo , Glicolipídeos/metabolismo , Metabolômica , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Glicolipídeos/química , Glicolipídeos/genética , Metabolômica/métodos , Mutação , Oxirredução , Peroxissomos/química , Peroxissomos/genética , Peroxissomos/metabolismo , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
Chemistry the worm's way: The nematode Pristionchus pacificus constructs elaborate small molecules from modified building blocks of primary metabolism, including an unusual xylopyranose-based nucleoside (see scheme). These compounds act as signaling molecules to control adult phenotypic plasticity and dauer development and provide examples of modular generation of structural diversity in metazoans.
Assuntos
Nematoides/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Ácidos Graxos/metabolismo , Metaboloma , Nucleosídeos/química , Nucleosídeos/metabolismo , Feromônios/química , Feromônios/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Xilose/análogos & derivados , Xilose/química , Xilose/metabolismoRESUMO
Surgical resection of tumours requires precisely locating and defining the margins between lesions and normal tissue. However, this is made difficult by irregular margin borders. Although molecularly targeted optical contrast agents can be used to define tumour margins during surgery in real time, the selectivity of the contrast agents is often limited by the target being expressed in both healthy and tumour tissues. Here, we show that AND-gate optical imaging probes that require the processing of two substrates by multiple tumour-specific enzymes produce a fluorescent signal with significantly improved specificity and sensitivity to tumour tissue. We evaluated the performance of the probes in mouse models of mammary tumours and of metastatic lung cancer, as well as during fluorescence-guided robotic surgery. Imaging probes that rely on multivariate activation to selectively target complex patterns of enzymatic activity should be useful in disease detection, treatment and monitoring.
Assuntos
Meios de Contraste/química , Cirurgia Assistida por Computador/métodos , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Fluorescência , Corantes Fluorescentes/química , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/cirurgia , Camundongos , Camundongos Endogâmicos BALB C , Imagem Óptica/métodos , Células RAW 264.7RESUMO
Optical contrast agents containing near-infrared (NIR) fluorophores are useful for visualizing biological landmarks, enzyme activities and biological processes in live animals and humans. Activatable (smart) quenched-fluorescent probes are sensors that become fluorescent after processing by an enzyme or in response to a physiological change (i.e., pH, ROS, etc.). Recently, there has been increased interest in developing activatable probes for research and clinical applications. This requires evaluation using in vivo animal models to gain insights into the pharmacodynamic and pharmacokinetic properties of a given probe. Important parameters to measure when evaluating quenched-fluorescent probes are signal brightness and signal-to-background ratios, which define the sensitivity and specificity of a probe. In this chapter, we discuss methods to evaluate activatable quenched-fluorescent probes in mouse models of cancer. Quantification of fluorescent signal intensity, calculation of tumor-to-background ratios, comparison of fluorescent activation in specific organ compartments, and fluorescence scanning of sectioned tissue will be discussed.
Assuntos
Corantes Fluorescentes , Neoplasias , Animais , Meios de Contraste , CamundongosRESUMO
Otitis media (OM) or middle ear infection is one of the most common diseases in young children around the world. The diagnosis of OM is currently performed using an otoscope to detect middle ear fluid and inflammatory changes manifested in the tympanic membrane. However, conventional otoscopy cannot visualize across the tympanic membrane or sample middle ear fluid. This can lead to low diagnostic certainty and overdiagnoses of OM. To improve the diagnosis of OM, we have developed a short-wave infrared (SWIR) otoscope in combination with a protease-cleavable biosensor, 6QC-ICG, which can facilitate the detection of inflammatory proteases in the middle ear with an increase in contrast. 6QC-ICG is a fluorescently quenched probe, which is activated in the presence of cysteine cathepsin proteases that are up-regulated in inflammatory immune cells. Using a preclinical model and custom-built SWIR otomicroscope in this proof-of-concept study, we successfully demonstrated the feasibility of robustly distinguishing inflamed ears from controls (p = 0.0006). The inflamed ears showed an overall signal-to-background ratio of 2.0 with a mean fluorescence of 81 ± 17 AU, while the control ear exhibited a mean fluorescence of 41 ± 11 AU. We envision that these fluorescently quenched probes in conjunction with SWIR imaging tools have the potential to be used as an alternate/adjunct tool for objective diagnosis of OM.
Assuntos
Otite Média com Derrame , Otite Média , Criança , Pré-Escolar , Orelha Média , Fluorescência , Humanos , Otite Média/diagnóstico , OtoscopiaRESUMO
PURPOSE: The reoperation rate for breast-conserving surgery is as high as 15-30% due to residual tumor in the surgical cavity after surgery. In vivo tumor-targeted optical molecular imaging may serve as a red-flag technique to improve intraoperative surgical margin assessment and to reduce reoperation rates. Cysteine cathepsins are overexpressed in most solid tumor types, including breast cancer. We developed a cathepsin-targeted, quenched fluorescent activity-based probe, VGT-309, and evaluated whether it could be used for tumor detection and image-guided surgery in syngeneic tumor-bearing mice. METHODS: Binding specificity of the developed probe was evaluated in vitro. Next, fluorescent imaging in BALB/c mice bearing a murine breast tumor was performed at different time points after VGT-309 administration. Biodistribution of VGT-309 after 24 h in tumor-bearing mice was compared to control mice. Image-guided surgery was performed at multiple time points tumors with different clinical fluorescent camera systems and followed by ex vivo analysis. RESULTS: The probe was specifically activated by cathepsins X, B/L, and S. Fluorescent imaging revealed an increased tumor-to-background contrast over time up to 15.1 24 h post probe injection. In addition, VGT-309 delineated tumor tissue during image-guided surgery with different optical fluorescent imaging camera systems. CONCLUSION: These results indicate that optical fluorescent molecular imaging using the cathepsin-targeted probe, VGT-309, may improve intraoperative tumor detection, which could translate to more complete tumor resection when coupled with commercially available surgical tools and techniques.
RESUMO
Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes <1% of the tissue margins. A quenched protease-activated fluorescent probe 6qcNIR, which produces a signal only in the protease-rich tumor microenvironment, was topically applied to 90 specimens ex vivo immediately following excision. "Puzzle-fit" analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence, resulting in sensitivities of 62%-78% and specificities of 92%-97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating en face images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing same-day reexcision when needed, reducing patient anxiety and improving compliance by expediting postsurgical specimen assessment. SIGNIFICANCE: A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment of margins during skin cancer excision, allowing same-day reexcision when needed.
Assuntos
Carcinoma Basocelular/cirurgia , Carcinoma de Células Escamosas/cirurgia , Procedimentos Cirúrgicos Dermatológicos/métodos , Imagem Óptica/métodos , Neoplasias Cutâneas/cirurgia , Cirurgia Assistida por Computador/métodos , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Queratinócitos/patologia , Masculino , Margens de Excisão , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
In the nematodes Caenorhabditis elegans and Pristionchus pacificus, a modular library of small molecules control behavior, lifespan, and development. However, little is known about the final steps of their biosynthesis, in which diverse building blocks from primary metabolism are attached to glycosides of the dideoxysugar ascarylose, the ascarosides. We combine metabolomic analysis of natural isolates of P. pacificus with genome-wide association mapping to identify a putative carboxylesterase, Ppa-uar-1, that is required for attachment of a pyrimidine-derived moiety in the biosynthesis of ubas#1, a major dauer pheromone component. Comparative metabolomic analysis of wild-type and Ppa-uar-1 mutants showed that Ppa-uar-1 is required specifically for the biosynthesis of ubas#1 and related metabolites. Heterologous expression of Ppa-UAR-1 in C. elegans yielded a non-endogenous ascaroside, whose structure confirmed that Ppa-uar-1 is involved in modification of a specific position in ascarosides. Our study demonstrates the utility of natural variation-based approaches for uncovering biosynthetic pathways.
Assuntos
Genômica , Metabolômica , Nematoides/genética , Nematoides/metabolismo , Feromônios/biossíntese , Feromônios/genética , Animais , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Feromônios/químicaRESUMO
The Precision Medicine Initiative aims to use advances in basic and clinical research to develop therapeutics that selectively target and kill cancer cells. Under the same doctrine of precision medicine, there is an equally important need to visualize these diseased cells to enable diagnosis, facilitate surgical resection, and monitor therapeutic response. Therefore, there is a great opportunity for chemists to develop chemically tractable probes that can image cancer in vivo. This review focuses on recent advances in the development of optical probes, as well as their current and future applications in the clinical management of cancer. The progress in probe development described here suggests that optical imaging is an important and rapidly developing field of study that encourages continued collaboration among chemists, biologists, and clinicians to further refine these tools for interventional surgical imaging, as well as for diagnostic and therapeutic applications.
Assuntos
Meios de Contraste/química , Corantes Fluorescentes/química , Neoplasias/diagnóstico , Imagem Óptica/métodos , Peptídeos/química , Medicina de Precisão/métodos , Bibliotecas de Moléculas Pequenas/química , Animais , Meios de Contraste/metabolismo , Meios de Contraste/farmacocinética , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacocinética , Humanos , Modelos Moleculares , Neoplasias/metabolismo , Neoplasias/patologia , Peptídeos/metabolismo , Peptídeos/farmacocinética , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacocinéticaRESUMO
We describe a new type of collective behavior in C. elegans nematodes, aggregation of starved L1 larvae. Shortly after hatching in the absence of food, L1 larvae arrest their development and disperse in search for food. In contrast, after two or more days without food, the worms change their behavior--they start to aggregate. The aggregation requires a small amount of ethanol or acetate in the environment. In the case of ethanol, it has to be metabolized, which requires functional alcohol dehydrogenase sodh-1. The resulting acetate is used in de novo fatty acid synthesis, and some of the newly made fatty acids are then derivatized to glycerophosphoethanolamides and released into the surrounding medium. We examined several other Caenorhabditis species and found an apparent correlation between propensity of starved L1s to aggregate and density dependence of their survival in starvation. Aggregation locally concentrates worms and may help the larvae to survive long starvation. This work demonstrates how presence of ethanol or acetate, relatively abundant small molecules in the environment, induces collective behavior in C. elegans associated with different survival strategies.